A substantial growth of the intestines of newborn animals takes place in the first one to three days after birth. For example, in newborn pigs who are nursed by the mother, there is a substantial growth, approximately eight to ten inches, of the intestines of the infant within the first few days after birth. In a large number of human newborns, who are not nursed by the mother but are placed on an infant's formula, this growth of the gastrointestinal tract during the first few days may not occur, and, as a result, the infant is predisposed to chronic intractable diarrhea which must be managed for a period of three or more months at considerable expense and discomfort to the infant.
The present invention is based upon the discovery that milk lactoferrin as a dietary ingredient promotes the growth of the gastrointestinal tract when added to infant formula or given separately from the formula and thus reduces the occurrence of chronic diarrhea and may assist in the management of short-gut syndrome and avoids, at least to some extent, chronic intractable diarrhea of the infant. The lactoferrin may be from a nonhuman animal or human source. The milk containing the lactoferrin should not be processed, such as by pasturization or the lactoferrin processed, extracted, or purified by a process which destroys the effectiveness of the lactoferrin.
Mammary secretions from goats, sheep, cows, and humans have been found to stimulate the proliferation of various cell lines growing in culture (1-3). When purified, the active factor was found to be Epidermal Growth Factor (EGF). None of the cell lines used for the bioassay of previous mitogenic factors exhibit polarity and none have brush border membranes.
In a search for factors in human colostrum which might stimulate enterocyte proliferation, we developed an assay for thymidine incorporation into DNA using harvested crypt cells from mature rat small intestine. Whole skimmed human colostrum stimulated a significant increase in thymidine incorporation into crypt cell DNA during a 60 minute period of incubation. When the protein with biological activity was purified to a single peak by sequential ion and gel chromatography, it was found to have the characteristics of lactoferrin. The mobility of SDS electrophoresis and electrofocusing was identical to that of standard lactoferrin. The protein was identical to these standards on double-diffusion immunologic testing. All available human lactoferrins stimulated thymidine uptake and all reacted with a lactoferrin polyclonal antibody. Human lactoferrin appears to be a potent activator of thymidine incorporation into DNA in incubated rat crypt cells, a biological activity not previously reported.
In addition, we found that EGF does not stimulate crypt cells. This finding supports our hypothesis that the response of 3T3 fibroblasts to EGF is not synonymous with a trophic effect on polarized intestinal epithelial cells.
Studies in the pig, dog, and rat indicate that the gastrointestinal tract matures more rapidly if the newborn animal is suckled (1-3). The nutritional significance of these observations lies in the principle that the structure of ingested protein may have biological significance beyond the dietary requirement for amino acids. Based on these observations, in vitro fibroblasts and other cell lines have been used to test for the presence of growth-promoting factors in milk. Mammary secretions from goats, sheep, cows, and humans have been found to stimulate the proliferation of various cell lines growing in culture (4-6). A portion of the activity in fibroblast culture can be attributed to EGF, a 6000-M.sub.r mitogen present in many mammalian secretions (7). Other factors with mitogenic activity, however, have been identified, e.g. polypeptides with M.sub.r of approximately 140,000 and 36,000(5).
None of the cell lines used for the bioassay of mitogenic factors exhibit polarity and none have brush-border membranes. Assuming that specificity may exist in intestinal cells, we developed a bioassay based on harvested rat crypt enterocytes. The bioassay works equally well with harvested pig crypt enterocytes. With this assay, we have confirmed the presence of mitogenic activity in human colostrum. Subsequently set forth herein are the details of the bioassay, the isolation of lactoferrin as a mitogenic dietary factor present in human and bovine milk, and compare the enterocyte assay with the fibroblast assay system used by Klagsbrun (5).
Although the mechanisms by which lactoferrin may stimulate the production of DNA in crypt cells have not been described, a similar protein, transferrin, is known to have a stimulating effect in a variety of cell lines. The two proteins, however, are immunologically distinct. Transferrin is an essential component of highly defined tissue culture media with a requirement of less than 10 .mu.g/ml for most cell lines. Whether this property of transferrin is attributable to the iron or to the apotransferrin protein has not been determined (19). In the cell lines studied thus far, an exclusive receptor is present for either lactoferrin or transferrin (20). Although transferrin and lactoferrin are not interchangeable, we have discovered that lactoferrin has a role parallel to that of transferrin in vitro and in vivo.
We have discovered that lactoferrin both human and animal stimulated thymidine incorporation into DNA by rat or pig crypt enterocytes. Human milk is known to stimulate thymidine uptake in a variety of fibroblast cell lines. The factors responsible for the initiation of mitosis have been identified in part. EGF was the first described and is the best known active factor in human milk. Receptors at the plasma membrane of 3T3 fibroblasts bind EGF (epidermal growth factor) and internalize it for subsequent nuclear binding. This mechanism requires 12 to 14 hours for completion when confluent fibroblast cultures are stimulated (16). In the assay with rat crypt cells, the DNA was harvested after only one hour of incubation. The short incubation may be one reason for the failure of EGF to stimulate enterocytes. The cells in the crypt cell assay may have been conditioned in vivo by EGF before they were harvested for the in vitro bioassay (17).
Klagsbrun and coworkers (18) have identified three factors in human milk which stimulate cell proliferation. The 3T3 cell line responds in vitro to whole human milk and to purified fractions. Klagsbrun's fractions I and II and EGF accounted for 5, 20, and 75%, respectively, of the 3T3 stimulation by human milk. Both larger fractions are broken down to smaller M.sub.r fractions under denaturing conditions. Fraction II is resolved by isoelectric focusing into two fractions with different pI. Based on the reported M.sub.r and PI, none of Klagsbrun's fractions appear to be intact lactoferrin. The cathodic protein in his factor II may be a fragment from intact lactoferrin, but the relative resistance of lactoferrin to proteolysis makes this possibility unlikely. Assays with 3T3 cells confirmed (data not shown) that fractions of human colostrum stimulate thymidine incorporation in this cell line. Human lactoferrin, however, does not promote growth in the 3T3 bioassay which is sensitive to EGF. The absence of sensitivity to lactoferrin in the 3T3 cell line explains why previous investigators have not observed its stimulation of thymidine incorporation in this fibroblast system.
Bovine lactoferrin purified by two different commercial processes is active in the crypt cell bioassay. This is in contract to the lack of stimulatory activity in cow's milk-based infant's formulas. The lactoferrin was from bovine colostrum and bovine mature milk and was acquired from Sigma Chemical Co., St. Louis, Mo.
The biological significance of lactoferrin-induced thymidine incorporation in rat and pig crypt cell DNA has not been elucidated previously. What is clear, however, is that lactoferrin is inactive in 3T3 cell lines which are sensitive to EGF (FIG. 9) and responsive to Klagsbrun's fractions I and II and that EGF is inactive in the crypt cell bioassay sensitive to lactoferrin (FIG. 1).